Summary: Chronic infection with hepatitis B virus (HBV) is a major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma (HCC) worldwide. Globally there are an estimated 240 million persons infected with HBV. In the United States, there are 1.25 million affected individuals and the epidemiology of the infection is changing due to immigration of persons from endemic regions. The natural history of chronic hepatitis B (CHB) also appears to be changing with an increasing prevalence of HBeAg negative chronic hepatitis B. Knowledge of the rate of progression between individuals with HBeAg positive and negative CHB is unknown. An equally important and related issue is the clinical assessment of disease severity in patients with CHB. Unfortunately, there are no good laboratory markers of disease severity. Liver biopsy is the accepted gold standard for assessing disease severity and cirrhosis but is costly, invasive, and associated with complications, which often limits patient acceptability as well as being subject to sampling error ranging from 15% to 20%. Non-invasive methods to assess disease severity are highly desirable for physicians caring for patients with CHB. Despite the availability of safe and effective oral nucleoside analogues for treatment of CHB, therapy remains problematic due to the need for prolonged therapy and limited effectiveness and tolerability of the alternate treatment, interferon. Clearance of hepatitis B surface antigen is the desired surrogate endpoint of therapy but is rarely achieved with current therapy. Identifying the optimal regimen, defining when to treat, for how long and when to stop therapy to achieve this endpoint are major unresolved issues. In addition, defining the best parameters to monitor patients both on and off therapy are not clear. Hypotheses/problems addressed: 1) Define the host, viral and environmental factors that determine the natural history and outcome of HBV infection. Hepatitis B e antigen seroconversion is an important milestone in the disease and is the treatment endpoint for patients who are HBeAg positive. The outcome of infection after HBeAg seroconversion is highly variable and little is known about the factors that affect outcome after HBeAg seroconversion. We explored whether two naturally occurring mutations (pre-core and basal core promoter mutations) could affect outcome after HBeAg seroconversion. We used pyrosequencing to quantify and longitudinally monitor the presence of naturally occurring mutations (pre-core and basal core promoter mutations) and correlated this with phenotype after HBeAg seroconversion. We demonstrated that the level of both pre-core and basal core promoter mutations correlated closely with HBV phenotype. This offers a means to predict phenotype after HBeAg seroconversion. Finally, we analyzed durability of spontaneous or treatment-related HBsAg loss. We observed that HBsAg was durable in 95% of patients after 9 years of follow-up and therefore represents the ideal endpoint of antiviral treatment. The Liver Diseases Branch is participating in a large multicenter study, the Hepatitis B Research Network, to define the natural history of HBeAg positive and negative chronic hepatitis B. This study has enrolled 2,000 patients at 13 North American sites. We recently published an analysis examining the prevalence of HBV-drug resistant (HBV-DR) variants among participants of the Hepatitis B Research Network (HBRN) using Sanger and next generation sequencing. Primary HBV-DR variants were detected by Sanger sequencing in (1.2%) participants: In a subset of 132 participants, NGS detected HBV-DR variants in a higher proportion of participants: 13.6%. Based on Sanger sequencing, the prevalence of primary HBV-DR variants is low (1.2%) among adults with CHB in US/Canada. This low prevalence of HBV-DR variants in patients with and without NUC treatment suggests transmission of these variants is uncommon. 2) Develop and evaluate novel, safer and more effective therapies for chronic viral hepatitis. Current therapy of CHB remains less than optimal. Relapse is common if treatment is discontinued after one year in the absence of HBsAg loss. Consequently, nucleos(t)ides must often be administered long-term or indefinitely. However, long-term use is associated with increased risk of side effects and higher costs. Therefore, the focus of current studies is to develop strategies to induce HBsAg loss (functional cure) to permit discontinuation of therapy and improve outcome of the infection. We are taking multiple approaches to this problem. The first approach is to discontinue therapy in patients receiving long-term nucleos(t)ide analogues. To address this question we have an ongoing prospective trial to withdraw patients from medication and observe for benefits (HBsAg loss) or adverse events (ALT flares, HBeAg seroreversion and fulminant hepatitis). A total of 15 patients have been withdrawn from therapy and the last patient completed the last follow-up visit August 2017. Analysis is ongoing. The second approach is to combine peginterferon alfa with tenofovir compared to tenofovir alone. This is being conducted under the auspices of the Hepatitis B Research Network and is a multicenter trial that enrolled 200 subjects and is ongoing. The third approach is to add peginterferon to ongoing long-term nucleos(t)ide analogue therapy for a period of 6 month. This trial will explore the mechanism of action of interferon during therapy of chronic hepatitis B. This trial has enrolled 13 subjects to date and is ongoing. 3) Elucidate the viral pathogenesis of HBV infection and mechanisms of anti-viral resistance The course of CHB is variable and affected by host, viral and environmental factors. We are investigating the role of pre-core and basic core promoter mutations on the outcome of chronic HBV infection after HBeAg serconversion. We hypothesize that elevation in HBV DNA levels are due to an increase in variant virus over wild type virus. We have quantified the proportion of pre-core and basic core promoter mutations relative to wild type virus among inactive carriers (HBV DNA <2000 IU/ml and normal ALT), indeterminate chronic HBV infection (HBV DNA >2000-<20,000 IU/ml and any ALT value) and HBeAg negative chronic hepatitis B (HBV DNA >20,000 IU/ml and ALT >2XULN). The results showed that the HBV phenotype was strongly related to the proportion of pre-core variant and ALT flares were more common when the proportion of either the pre-core and basic core promoter variants increased in the serum. The level of hepatitis B virus among the infected population is broad and ranges 9 log10 IU/ml. The cause for this is unknown. We are performing whole genome sequencing from patients that span the range of viral loads and patients with atypical HBV phenotype to identify mutations that may affect viral replication. Novel mutations identified in the sequence analysis will be engineered into a laboratory replicative construct (wildtype adwR9), using site directed mutagenesis. Replication competence of these mutant constructs will then be assessed after transient transfection of HuH-7 cells. Comparison of replication levels (measured by HBV DNA and HBsAg quantitation) will be made with wildtype adwR9 to determine whether these mutations upregulate or downregulate HBV replication. A manuscript is in preparation. We analyzed the kinetics of HBsAg loss using a quantitative HBsAg assay among both treated and untreated patients to understand the mechanisms for clearance of HBsAg-the viral protein associated with chronic infection. This work was presented as an oral presentation at DDW. A manuscript is in preparation. Finally, we have performed an unbiased microRNA analysis and correlated this with HBV phenotype. Analysis is ongoing.